Improvements in the management and treatment of debilitated medical and surgical patients have been accompanied by an unfortunate increase in the number of life-threatening infections due to pathogenic and opportunistic fungi (McNeil et al., Clin. Infect. Dis. 33:641-47, 2001). AIDS, cancer chemotherapy and high dose corticosteroid treatment have all contributed to the increasing number of immunocompromised individuals. Many immunocompromised subjects develop opportunistic infections with saprophytic filamentous fungi, such as Aspergillus species, which are found in the environment and were originally considered to be of low virulence (Latge, Clin. Microbiol. Rev. 12:310-50, 1999). These infections are often fulminant and fatal in immunocompromised patients. For example, pulmonary and cerebral aspergillosis have mortality rates of 86 and 99%, respectively, even when adequately treated (Denning, Clin. Infect. Dis. 23:608-14, 1996).
The advent of new, specific antifungal drugs and treatment regimes has improved the prospects for management of aspergillosis. However, diagnosis remains difficult, and early initiation of appropriate antifungal therapy is critical in reducing mortality rates in immunocompromised patients (Einsele et al., J. Clin. Microbiol. 35:1353-60, 1997). Hence rapid diagnostic assays are needed to improve treatment outcomes.
Aspergillus is one of the primary pathogens which cause systemic fungal infection treated in hospitals. It usually affects subjects who have had organ transplants, acute leukemias and burns, and can be rapidly fatal if not diagnosed quickly. There are over 150 species of Aspergillus present in the soil, air and water, hence accurate detection of important species of Aspergillus is often complex and difficult.
Diagnosis of fungal infections is typically made by isolation of the infecting organism in culture, by serologic assays, or through histopathologic examination of tissue (Hamilton, Med. Mycol. 36:351-64, 1998). Isolation of Aspergillus species in culture can require several days for adequate growth and sporulation to occur, delaying appropriate drug therapy, and a positive culture may represent benign colonization rather than true invasion or infection (de Repentigny, Clin. Infect. Dis. 14:S11-22, 1992). When histopathology is performed on tissue sections, the morphological similarities of the various Aspergillus species can make definitive species identification difficult.
Alternatively, serological tests can be used to diagnose fungal infections, but most such tests lack the desired sensitivity and/or specificity for a confident diagnosis. Serologic tests on a single serum sample to detect circulating fungal antigens may be inconclusive, and antibody production in the immunocompromised patient population most at risk for invasive aspergillosis is often variable and an unreliable diagnostic indicator (Morrison and Lindsley, Fungal Pathogenesis: Principles and Practice, Marcel Dekker, Inc., 667-716, 2001).
Current serological assays do not identify Aspergillus to the species level, and are both time-consuming and expensive. In addition, Aspergillus terreus has been shown to be resistant to amphotericin B, the most commonly prescribed drug for treating invasive aspergillosis. Aspergillus fumigatus has been reported to develop resistance to itraconazole. Thus, there remains a need for a rapid test to identify aspergilli to the species level, to help assist in the selection of appropriate drugs for the treatment of clinical Aspergillus infections. Fungal species identification may also be important for detecting organisms in the environment that may be potentially pathogenic, for example to an immunocompromised person who is exposed to that environment.
PCR-based methods of detection, which show promise as rapid techniques to diagnose infections, have been used in the identification of DNA from Candida and Aspergillus species. However, most of these tests are only genus specific, and are unable to specifically identify many clinically and environmentally important Aspergillus species.
Unique internal transcribed sequence 2 (ITS2) coding regions have been used to develop nucleic acid probes for several different species of Aspergillus (A. flavus, A. fumigatus, A. niger, A. terreus, and A. nidulans), as disclosed in U.S. Pat. No. 6,372,430, which is incorporated by reference.